Effect of PPARy Ligands on Alcohol-Induced Alveolar Macrophage Oxidative Stress

描述(由申请人提供)：长期酗酒会增加患者患急性呼吸窘迫综合症(ARDS)和呼吸道感染的风险。在肺泡巨噬细胞(AM)中，NADPH氧化酶(NOX)1、NOX2和NOX4是活性氧(ROS)的重要来源，而NOX2是吞噬后参与杀死微生物的呼吸爆发所必需的。然而，过量的ROS产生会抑制吞噬作用。长期摄入酒精会增加NOx酶水平，导致AM氧化应激和功能障碍。这些酒精诱导的排列紊乱可以被过氧化体增殖物激活的受体γ(PPAR？)逆转。配体，如吡格列酮和罗格列酮。在这些研究中，PI将通过研究microRNAs(MiRs)来阐明调控酒精诱导AM Nox表达和活性的分子机制：与Nox1相关的miR-1264，与NOX2相关的miR-107，以及与NOX4相关的miRs-363和-92a/b(Aim 1)。那么，PI将如何审查PPAR？配体减弱这些MIR以逆转酒精介导的AM NOX1、NOX2和NOX4的表达、氧化应激和吞噬功能受损(目标2)。这些假说将通过使用慢性酒精消费的小鼠模型、体外乙醇暴露的小鼠AM细胞系MH-S和从人类受试者分离的AM来验证。本申请中概述的研究的目标是证明以PPAR？为改善酒精引起的AM功能障碍提供了一种新的治疗方法。如果成功，这些研究可能会为未来的临床研究奠定基础，从而对有酗酒史的患者的管理产生相当大的翻译影响。PI对酒精研究的关注始于她的博士前论文项目，该项目调查慢性酒精滥用在肝脏中的有害影响，重点是ROS和炎症介质参与改变肝脏内皮细胞和巨噬细胞功能的潜在机制。在布朗博士和哈特博士的实验室进行博士后培训期间，她在众多分子生物学技术和慢性酒精摄入动物模型方面获得了额外的专业知识。在拟议项目的K99指导阶段，申请者将进一步扩大她的技能库，接受以下方面的动手培训：a)在小鼠模型的呼吸道进行和表征肺炎克雷伯菌细菌挑战的技术，以评估体内肺泡巨噬细胞的吞噬作用，b)直接传递PPAR的方法？使用鼻腔给药对小鼠模型的肺部进行配基治疗，以及c)开发和管理基因敲除和转基因小鼠群体所需的技能。在拟议项目期的头两年，将在导师实验室的协助和培训下，通过执行拟议的研究来获得这些技能。这些研究的重点将允许候选人在慢性酒精摄入的背景下自然地扩展对MIR的调节的兴趣。预计她在这一领域的额外专业知识将自然而然地促进她从导师那里越来越独立地进入R00独立阶段。在拟议的奖项期间，除了教授现代分子生物学技术和生物统计学方法的课程外，PI还将通过参加与负责任的研究、实验室管理和教师职业发展相关的研讨会和活动，获得对她作为独立研究调查员的职业发展至关重要的非实验室技能。K99/R00助学金的支持将为PI提供一个极好的机会来扩展和巩固她的实验和实验室技能，并支持她成为一名独立调查员并在学术机构获得教员职位的职业目标。她实现这些目标的可能性得到了来自知名研究人员的有计划的指导、埃默里大学酒精和肺部生物学中心内丰富的科学机会，以及一个探索一种重要和临床相关病理生理疾病的新机制的假设驱动的应用程序的支持。拟议的方案将允许私人投资建立她的出版记录，为随后的赠款申请收集关键的初步数据，在全国会议上提交研究结果，并获得手稿写作和赠款准备方面的经验。因此，这份K99/R00申请提供了一个极好的机会来推动一名有才华和有前途的研究员的职业生涯。

DESCRIPTION (provided by applicant): Chronic alcohol abuse increases patients' risk of developing Acute Respiratory Distress Syndrome (ARDS) and respiratory infections. In alveolar macrophages (AMs), NADPH oxidase (Nox) 1, Nox2, and Nox4 are critical sources of reactive oxygen species (ROS), and Nox2 is essential for the respiratory burst involved in killing microbes after phagocytosis. However, excessive ROS production suppresses phagocytosis. Chronic alcohol ingestion increases Nox enzyme levels, leading to AM oxidative stress and dysfunction. These alcohol-induced derangements can be reversed by treatment with peroxisome proliferator-activated receptor gamma (PPAR?) ligands, such as pioglitazone and rosiglitazone. In these studies, the PI will elucidate the molecular mechanisms that modulate alcohol-induced AM Nox expression and activity by studying microRNAs (miRs): Nox1-related miR-1264, Nox2-related miR-107, and Nox4-related miRs-363 and -92a/b (Aim 1). Then, the PI will examine how PPAR? ligands attenuate these miRs to reverse alcohol-mediated AM Nox1, Nox2, and Nox4 expression, oxidative stress and compromised phagocytosis (Aim 2). These hypotheses will be investigated by using a mouse model of chronic alcohol consumption, an in vitro ethanol exposed mouse AM cell line, MH-S, and AMs isolated from human subjects. The objective of the studies outlined in this application is to demonstrate that targeting PPAR? constitutes a novel therapeutic approach to ameliorate alcohol-induced AM dysfunction. If successful, these investigations could have considerable translational impact on the management of patients with a history of alcohol abuse by setting the stage for future clinical studies. The PI's focus in alcohol research began during her pre-doctoral dissertation project investigating the detrimental effects of chronic alcohol abuse in the liver, focusing on mechanisms underlying the participation of ROS and inflammatory mediators that alter liver endothelial cell and macrophage function. During post- doctoral training in the laboratories of Drs. Brown and Hart, she acquired additional expertise with numerous molecular biology techniques and with animal models of chronic alcohol ingestion. During the K99 mentored phase of the proposed project, the applicant will further expand her repertoire of skills by receiving hands-on training in: a) techniques to perform and characterize Klebsiella pneumonia bacterial challenges in the airways of mouse models to assess alveolar macrophage phagocytosis in vivo, b) methods to directly deliver PPAR? ligand therapeutics to the lungs of mouse models using intranasal administration, and c) skills required to develop and manage colonies of knockout and transgenic mice. These skills will be acquired through the execution of the proposed studies with the assistance and training of the mentors' labs during the first two years of the proposed project period. The focus of these studies will permit a natural extension of the candidate's interest in the regulation of miRs in the context of chronic alcohol ingestion. t is anticipated that her additional expertise in this area will naturally promote her growing independence from her mentors into the R00 independent phase. During the proposed award, in addition to didactic courses in current molecular biology techniques and biostatistical methods, the PI will gain non-laboratory skills important for her career development as an independent research investigator by participating in seminars and activities related to the responsible conduct of research, laboratory management, and faculty career development. Support from this K99/R00 grant will provide the PI an outstanding opportunity to expand and consolidate her experimental and laboratory skills and support her career goal to become an independent investigator and obtain a faculty position at an academic institution. The likelihood that she will achieve these goals is supported by planned mentorship from well-established investigators, the abundant scientific opportunities within the Emory University Alcohol and Lung Biology Center, and a hypothesis-driven application exploring novel mechanisms of an important and clinically relevant pathophysiological disorder. The proposed program will permit the PI to build her publication record, collect critical preliminary data for subsequent grant applications, present research findings at national meetings, and gain experience in manuscript writing and grant preparation. Thus, this K99/R00 application provides an excellent opportunity to advance the career of a talented and promising investigator.